Thyagarajan B, McCormick-Graham M, Romero D P, Campbell C
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA.
Nucleic Acids Res. 1996 Oct 15;24(20):4084-91. doi: 10.1093/nar/24.20.4084.
Homologous DNA recombination levels were measured in normal and spontaneously immortalized murine and human fibroblasts, and in a number of primate and murine established fibroblast cell lines. Immortal cell lines and tumor-derived clones homologously recombined extrachromosomal plasmid substrates at frequencies approximately 100-fold higher than did normal cells. To further explore the mechanism responsible for this phenotype, homologous recombination frequency was measured using nuclear extracts derived from normal and immortalized murine and human fibroblasts. Extracts prepared from immortal cells catalyzed high levels of homologous recombination, whereas very little recombination activity was detected in extracts prepared from normal fibroblasts. Similarly, only extracts derived from immortal cells contained strand-transferase activity as measured by the recently described pairing-on-membrane assay. Mixing experiments indicated that a recombination enhancing factor or factors present in immortal cells, rather than a recombination inhibitor in normal cells, was responsible for the enhanced homologous recombination activity observed using extracts derived from the former.
在正常的、自发永生化的小鼠和人类成纤维细胞，以及一些灵长类和小鼠的已建立的成纤维细胞系中，测定了同源DNA重组水平。永生化细胞系和肿瘤衍生克隆对染色体外质粒底物进行同源重组的频率比正常细胞高约100倍。为了进一步探究导致这种表型的机制，使用从正常的和永生化的小鼠及人类成纤维细胞中提取的核提取物来测定同源重组频率。从永生化细胞制备的提取物催化高水平的同源重组，而从正常成纤维细胞制备的提取物中检测到的重组活性非常低。同样，通过最近描述的膜上配对试验测定，只有从永生化细胞中提取的提取物含有链转移酶活性。混合实验表明，永生化细胞中存在的一种或多种重组增强因子，而非正常细胞中的重组抑制剂，是导致使用从前者提取的提取物观察到的同源重组活性增强的原因。
